本文選題：乳腺 + 干細胞�。� 參考：《第一軍醫(yī)大學》2007年碩士論文

【摘要】： 背景和目的 哺乳動物的乳腺是一種復(fù)管泡狀腺，具有分泌乳汁的功能；是動物出生后唯一可以多次重復(fù)再生的器官。根據(jù)乳腺發(fā)育的特征，嚙齒類動物乳腺大致可以分為六個發(fā)育期：胚胎期、青春期、性成熟期、孕期、哺乳期和退化期。 在乳腺細胞分化過程中，往往由于高度分化而完全失去再生能力，最終導(dǎo)致細胞的衰老死亡；乳腺在發(fā)育進化過程中為了補償這一不足，保留了一部分未分化的原始細胞，稱為乳腺干細胞。 乳腺細胞的增殖和分化始終貫穿于乳腺發(fā)育及泌乳全過程，人們在細胞水平上進行了大量的乳腺上皮細胞增殖及分化的研究。這些研究揭示乳腺干細胞再生乳腺和分化異常導(dǎo)致乳腺癌變的機制，為乳腺腫瘤和癌癥預(yù)防及治療提供理論依據(jù)，為研究器官形成和細胞分化機理提供合適的模型。 乳腺干細胞研究的標記蛋白主要有CK5，Sca-1，α6-integrin，MUC~-／ESA~+以及CK19。 合適的乳腺干細胞特殊標記物的討論仍在進行。在各報道中所確定的鑒別乳腺干細胞的標記物不同，這是值得重視和進一步研究的現(xiàn)象。這種不同可能有多方面的原因：1．材料的來源不同；2．體內(nèi)和體外培養(yǎng)研究方法的不同；3．乳腺干細胞及其微環(huán)境隨著個體的發(fā)育也處于發(fā)育變化過程中；4．乳腺干細胞在建立復(fù)雜的調(diào)控機制、信號轉(zhuǎn)導(dǎo)系統(tǒng)和特殊功能過程中表達一系列相關(guān)的蛋白。 我們選取青春期、性成熟期、孕期、哺乳期的大鼠乳腺組織進行形態(tài)學研究，運用免疫熒光和免疫酶標記以及Western免疫印跡法，觀察Sca-1，CD24，Muc1在大鼠乳腺發(fā)育四個特殊時期的表達，研究這三種細胞標記物的分布，以探討乳腺干細胞的增殖和分化。 方法 1．乳腺組織石蠟切片的準備：6 w(體質(zhì)量180～220g)、9 w(體質(zhì)量210～230g)、孕15 d(體質(zhì)量350～380g)、產(chǎn)后3 d(體質(zhì)量330～350g)，每個時期1組，每組12只，乳腺組織塊大小為1.0 cm×1.0 cm×0.5 cm，制成4μm厚切片。 2．HE染色：研究乳腺發(fā)育四個時期的形態(tài)和小亮細胞的形態(tài)分布。 3．免疫組化酶標記：研究Sca-1，CD24和Muc1在乳腺發(fā)育四個時期的抗原陽性情況。 4．免疫熒光單標記：研究Sca-1，CD24，Muc1在乳腺發(fā)育四個時期的抗原陽性情況。 5．Western免疫印跡法：取6 w、9 w、孕15 d、產(chǎn)后3 d的SPF級雌性大鼠，每個時期1組，每組3只，檢測Sca-1，CD24，Muc1在乳腺發(fā)育四個時期的各自含量。 6．免疫熒光雙標記：Sca-1分別和CD24以及Muc1標記，觀察它們在四個時期的雙陽性情況。 結(jié)果 1．HE染色結(jié)果 6 w雌性大鼠乳腺末端主要是端芽結(jié)構(gòu)；9 w雌性大鼠乳腺主要是腺小葉。在孕15 d乳腺組織中，乳腺的中、次級導(dǎo)管分支大量增加，腺泡迅速發(fā)育且數(shù)量增多。在產(chǎn)后3 d的乳腺組織中，乳腺小葉充滿分泌腺泡，腺泡上皮的形態(tài)多樣，細胞內(nèi)出現(xiàn)較多空泡，在細胞核與細胞的游離面之間的胞質(zhì)中空泡特別多，小葉內(nèi)導(dǎo)管明顯可見，結(jié)締組織中的血管較多，小葉間隔很薄。 在6 w乳腺組織中，小亮細胞大多是數(shù)個或成群地分布，常位于乳腺分支導(dǎo)管及末端芽的基底部；在9 w乳腺組織中，小亮細胞分布于導(dǎo)管及腺泡的基底膜上，也部分位于肌上皮與腺上皮細胞之間；在孕15 d乳腺組織中，小亮細胞分布于小葉導(dǎo)管中；在產(chǎn)后3 d乳腺組織中，小亮細胞大多分布在分支導(dǎo)管中。 2．免疫組化酶標記結(jié)果 Sca-1~+細胞分別位于6 w雌性大鼠乳腺的脂肪細胞之間和腺泡周圍；9 w雌性大鼠乳腺脂肪細胞之間和腺泡周圍；孕15 d雌性大鼠乳腺脂肪墊的脂肪細胞之間表達；以及產(chǎn)后3 d雌性大鼠乳腺的脂肪細胞之間，輸乳管和乳腺脂肪墊中。 CD24~+細胞分別位于6 w雌性大鼠乳腺脂肪細胞之間；9 w雌性大鼠乳腺脂肪墊中；在孕15 d雌性大鼠乳腺腺泡周圍；在產(chǎn)后3 d雌性大鼠乳腺的葉間導(dǎo)管。 Muc1~+細胞分別位于6w雌性大鼠乳腺的脂肪細胞之間，孕15 d雌性大鼠乳腺脂肪墊中；以及在產(chǎn)后3 d雌性大鼠乳腺葉間導(dǎo)管。 3．免疫熒光單標記結(jié)果 9 w雌性大鼠葉間導(dǎo)管分別有Sca-1~+；CD24~+和Muc1~+細胞；產(chǎn)后3 d雌性大鼠母鼠乳腺分支導(dǎo)管分別有Sca-1~+；CD24~+和Muc1~+細胞。 4．Western免疫印跡法結(jié)果 Sca-1在6 w和9 w乳腺中的含量沒太大變化，產(chǎn)后3 d大鼠乳腺的Sca-1含量比孕15 d的少，活動期間的2個乳腺發(fā)育時期的Sca-1含量比靜止期的含量少。CD24在大鼠乳腺發(fā)育前6w，9 w和孕15 d三個時期含量沒太大變化，而在產(chǎn)后3 d大鼠乳腺CD24含量變少。Muc1在乳腺發(fā)育活動期的2個時期含量比靜止期的2個時期高。 5．免疫免疫熒光雙標記結(jié)果 6 w雌性大鼠乳腺脂肪墊；9 w雌性大鼠乳腺脂肪細胞間；孕15 d雌性大鼠乳腺脂肪細胞間；以及產(chǎn)后3 d雌性大鼠乳腺脂肪細胞間均發(fā)現(xiàn)有Sca-1和CD24雙陽性細胞。 6 w雌性大鼠乳腺腺泡周圍和分支導(dǎo)管；9 w雌性大鼠乳腺腺泡周圍；孕15d雌性大鼠乳腺脂肪細胞之間以及產(chǎn)后3 d雌性大鼠乳腺分支導(dǎo)管存在Muc1和Sca-1雙陽性細胞。 結(jié)論 1．活動期的乳腺上皮細胞在乳導(dǎo)管基質(zhì)中廣泛增殖，乳腺中、次級導(dǎo)管分支增加；腺泡迅速發(fā)育，，數(shù)量增多且胞體變大。與靜止期乳腺相比，這些明顯變化應(yīng)該是懷孕期間多種激素的作用結(jié)果；4個時期的生長增殖部位都出現(xiàn)小亮細胞。 2．部分Sca-1陽性細胞可能代表一類腔祖上皮細胞，而且在不同時期含量是有變化的。 3．關(guān)于乳腺干細胞的表面標志物在乳腺發(fā)育過程中可能有變化：(1)標志物性質(zhì)的變化，即有不同標志物的保留或更替；(2)標志物表達量的變化。實驗結(jié)果顯示CD24在前三個時期含量沒變化，在產(chǎn)后3 d發(fā)生變化可能是對稱分裂發(fā)生的比較多，也可能是細胞橫向分化，使得這個時期干細胞含量降低。 4．Muc1在分泌乳汁的腺泡周圍和導(dǎo)管內(nèi)側(cè)以及脂肪等多個部位表達，Muc1在乳腺發(fā)育活動期的2個時期含量比靜止期含量高，它可能是成熟腔上皮細胞的標志。 5．在乳腺發(fā)育時期，乳腺細胞(導(dǎo)管上皮細胞、腺泡上皮細胞、肌上皮細胞)大量擴增的主要來源有兩種可能，可能Sca-1~+細胞具有雙潛能，部分Sca-1~+細胞代表一類雙潛能祖細胞。
[Abstract]:Background and Purpose






The mammary gland of mammal is a reconstituted alveolar gland with the function of secretion of milk ;
Animal mammary glands can be divided into six developmental stages : embryonic stage , adolescence , sexual maturity , pregnancy , lactation and degeneration .






In the process of mammary gland cell differentiation , the regeneration ability is often completely lost due to high differentiation , which leads to cell aging and death ;
In order to compensate for this deficiency in the course of development and evolution of the mammary gland , a portion of the undifferentiated primordial cells , referred to as mammary stem cells , are retained .






The proliferation and differentiation of mammary gland cells throughout the whole process of mammary gland development and lactation have been carried out in the whole process of mammary gland development and lactation .






There were CK5 , Sca - 1 , 偽6 - integrin , MUC ~ - / ESA ~ + and CK19 .






The discussion of a suitable marker for breast stem cells is still ongoing . This is a phenomenon that deserves attention and further research in the identification of markers for differentiation of breast stem cells identified in each report . This difference may have many reasons : 1 . the source of the material is different ;
2 . Different methods were developed in vivo and in vitro ;
3 . The mammary stem cells and their microenvironment are also in the process of development with the development of the individual ;
4 . Breast stem cells express a series of related proteins in the establishment of complex regulatory mechanisms , signal transduction systems and special functions .






In order to investigate the expression of Sca - 1 , CD24 and Muc1 during the four special periods of mammary gland development in rats , we observed the expression of Sca - 1 , CD24 and Muc1 in the rat mammary gland during the four special periods of mammary gland development , and studied the proliferation and differentiation of mammary stem cells .






method






1 . Preparation of paraffin section of breast tissue : 6 w ( body mass 180 ~ 220g ) , 9 w ( body mass 210 ~ 230g ) , pregnancy 15 days ( body mass 350 ~ 380g ) , postpartum 3 days ( body mass 330 ~ 350g ) , each time period 1 group , each group 12 , breast tissue mass size 1.0 cm 脳 1.0 cm 脳 0.5 cm , and made into 4 渭m thick section .






2 . HE staining : The morphology of four stages of mammary gland development and the morphological distribution of small bright cells were studied .






3 . Immunohistochemical staining was used to study the antigen - positive conditions of Sca - 1 , CD24 and Muc1 in four stages of mammary gland development .






4 . Immunofluorescent single labeling : study the antigen - positive condition of Sca - 1 , CD24 and Muc1 in four stages of mammary gland development .






5 . Western blot : SPF female rats with 6 w , 9 w , 15 d and 3 d postpartum were divided into 1 group , 3 in each group , and the contents of Sca - 1 , CD24 and Muc1 in four stages of mammary gland development were detected .






6 . Immunofluorescent double labeling : Sca - 1 and CD24 and Muc1 respectively were used to observe their double positive conditions in four periods .






Results






1 . HE Staining Results






The end - bud structure of the mammary gland of 6 w female rats was mainly the end bud structure .
The mammary gland of 9 w female rats is mainly glandular lobules . In the mammary gland tissue of 15 days after pregnancy , the branches of the secondary ducts increase and the number of acinar cells grow rapidly . In the mammary gland tissue of 3 days postpartum , there are many vacuoles in the mammary gland and the acinar cells , and there are more vacuoles in the cytoplasm of the nucleus and the free surface of the cells .






in 6 w breast tissue , most of that small bright cells are distribute in groups or groups , often located at the base of the breast branch catheter and the terminal bud ;
In 9 w breast tissue , the small bright cells were distributed on the basement membrane of catheter and acinar , and were also located between myoepithelial and glandular epithelial cells .
In the 15 d breast tissue , the small bright cells were distributed in the lobular duct .
In the postpartum 3 d breast tissue , the small bright cells are mostly distributed in the branch ducts .






2 . Immunohistochemical staining results






Sca - 1 ~ + cells were located between the fat cells and the acinar cells of the mammary gland of 6 w female rats .
9 w female rat mammary fat cells and around the acinar ;
The fat cells of mammary fat pad were expressed in 15 d female rats .
and postpartum 3 d female rat mammary gland fat cells , between the milk delivery tube and the breast fat pad .






CD24 ~ + cells were located between the mammary fat cells of 6 w female rats .
9 w female rat mammary fat pad ;
In the female rats of the 15th day of pregnancy , the mammary glands of the female rats were observed ;
Interleaf catheters in the mammary gland of female rat 3 d postpartum .






Muc1 ~ + cells were located in the fat cells of the mammary gland of 6w female rats , and in the mammary fat pad of 15 d female rats .
and in the postpartum 3 d female rat mammary gland duct .






3 . Immunofluorescence Single Sign Results






There were Sca - 1 ~ + in the leaves of 9 w female rats .
CD24 ~ + and Muc1 ~ + cells ;
Sca - 1 ~ + was found in the mammary gland branch ducts of female rats in 3 d postpartum .
CD24 ~ + and Muc1 ~ + cells .






4 . Western blot results






The content of Sca - 1 in the mammary glands of 6 w and 9 w was not significantly changed . The content of Sca - 1 in the mammary gland of 3 d rats was less than that of pregnant 15 d . The content of Sca - 1 in the mammary gland of rats was less than that in the rest period .






5 . Immune immunofluorescence double labeling results






6 w female rat mammary gland fat pad ;
9 w female rat mammary fat cells ;
The fat cells of mammary gland were observed in 15 d female rats .
Sca - 1 and CD24 - positive cells were found in fat cells of mammary gland in 3 d female rats .






6 w female rat mammary gland bubble around and branch catheter ;
9 w female rat mammary gland acinar ;
Muc1 and Sca - 1 double positive cells were present in the mammary gland branch ducts between 15 d female rat mammary fat cells and postpartum 3 d female rats .






Conclusion






1 . The mammary epithelial cells in the active period were proliferated in the matrix of the milk catheter , and the branches of the secondary ducts increased in the breast .
In contrast , these changes should be the result of multiple hormones during pregnancy .
Small bright cells appeared in the growth and multiplication sites in 4 periods .






2 . Some Sca - 1 - positive cells may represent a class of luminal epithelial cells and varied during different periods .






3 . There may be changes in the surface markers of breast stem cells in the mammary gland development : ( 1 ) changes in marker properties , i.e . retention or replacement of different markers ;
( 2 ) The results showed that CD24 was not changed during the first three stages , and the changes in 3 d after the postpartum period could be more or more likely to be a symmetric division , but also the lateral differentiation of the cells , so that the content of stem cells decreased during this period .






4 . Muc1 is expressed in the periphery of the gland of the secretion milk and the inner side of the catheter , as well as the fat and so on . Muc1 is higher than the rest period in 2 periods of the mammary gland development activity period , which may be the hallmark of mature luminal epithelial cells .






5 . There are two main sources of proliferation of mammary gland cells ( ductal epithelial cells , acinar epithelial cells , myoepithelial cells ) during the development of mammary glands . It is possible that Sca - 1 ~ + cells have dual potential , and some Sca - 1 ~ + cells represent a class of double - potential progenitor cells .

【學位授予單位】：第一軍醫(yī)大學
【學位級別】：碩士
【學位授予年份】：2007
【分類號】：R329

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4 張s

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